Some technical problems for the adequate analysis of the
expression of cloned cDNA are the lack of functional assays and/or
specific antibodies (Ab) to the proteins produced. To overcome this
difficulty fusion proteins where a known peptide is fused to the
expression product have been described. In addition to the Flag-Tag,
His-tag and GST-tag are both widely used.

The flag peptide that was first used was a 11-amino-acid leader
peptide of the gene-10 product from bacteriophage T7 fused at the
amino-terminus of GAL4 (yeast transcription factor). At the time,
there were no anti-GAL4 Ab commercially available (1), so a fusion
protein with an epitope recognized by a commercially available
antibody was prepared. The most widely used hydrophilic octapeptide
now is DYKDDDDK (2) though recent studies suggest that a shorter
peptide, DYKD, can be recognized with almost the same affinity by the
M1 monoclonal antibody (3). Also, new tag sequences have been
described for other monoclonal antibodies. The peptide MDFKDDDDK is
recognized by M5 and MDYKAFDNL recognized by M2 (4). The binding
reaction is also dependent on Calcium, so proteins can frequently be
eluted from an affinity matrix by EDTA containing buffer (16). The
beauty of this system is that it allows for the tag to be placed at
either the amino-terminus (5) carboxy-terminus (6), or in association
with other tags (7). It will not usually interfere with the fusion
protein expression, proteolytic maturation or activity (8, 17). So
far, even if the tag is placed in the MHC class I molecule, it will
not interfere with either alloantibody recognition or cytotoxic T
cell-MHC interactions (9).

The system provides immediate advantages such as purifying a
fusion protein by anti-Flag affinity chromatography (2), or in
one-step (3) and two-step (10) procedures and being able to use most
of the immunological techniques that involve monoclonal antibody
(ELISA, Western blot, FACS, immunohistochemistry, etc.). The most
recent uses of this technique are in transfection of eucaryotic cells
(11), mammalian cells (12), insect cells (6) and even transgenic
systems (13). Even more exciting is the idea that this system may
help solve one of the mayor problems in adenovirus gene transfer by
targeting the cells with the use of bispecific antibodies. These
bispecific antibodies bind to the flag peptide as well as to some
specific receptor on the cell surface, for example the alpha v
integrin in Wickham's experiment (14).

A mayor disadvantage of this system has been recently described.
This tag system depends on specific detection of flag fusion proteins
and no cross-reactivity to cellular proteins. Schafer et al.(15) have
just isolated a rat cDNA clone coding for a new isoform of a beta
phosphatase by screening a rat brain expression library with
monoclonal antibody M2. It seems that five out of the eight amino
acid residues in a sequence motif were identical. Even though this is
a problem to consider, the FlAG-tag system is a powerful tool that
has to be evaluated whenever Ab are not available to detect a fusion
protein.